Peptide inhibitors of HIV entry

ABSTRACT

Described herein are chimeric peptides comprising a soluble trimeric coiled-coil and all or a portion of the N-peptide region of HIV gp41. These molecules are stable, trimeric coiled-coils that inhibit HIV entry into cells, such as human cells. Such peptides can be further assessed to demonstrate their ability to serve as potent anti-HIV therapeutic molecules and thus, as therapeutic molecules or drugs.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US01/29637, which designated the United States and was filed Sep.21, 2001, published in English, which is a continuation of U.S.application Ser. No. 09/668,072, filed Sep. 22, 2000 now U.S. Pat. No.6,747,126 which is a continuation-in-part of U.S. application Ser. No.09/364,497, filed Jul. 30, 1999 now U.S. Pat. No. 6,818,740, and whichclaims the benefit of U.S. Provisional Application No. 60/132,295, filedMay 3, 1999, U.S. Provisional Application Ser. No. 60/101,058, filedSep. 18, 1998, U.S. Provisional Application No. 60/100,265, filed Sep.14, 1998, and U.S. Provisional Application No. 60/094,676, filed Jul.30, 1998.

This application is also a continuation of U.S. application Ser. No.09/746,742, filed on Dec. 21, 2000 now U.S. Pat. No. 6,841,657, which isa continuation of International Application No. PCT/US99/17351, whichdesignated the United States and was filed Jul. 30, 1999, published inEnglish, and which claims the benefit of U.S. Provisional ApplicationNo. 60/132,295, filed May 3, 1999, U.S. Provisional Application No.60/101,058, filed Sep. 18, 1998, U.S. Provisional Application No.60/100,265, filed Sep. 14, 1998, and U.S. Provisional Application No.60/094,676, filed Jul. 30, 1998.

This application is also a continuation of U.S. application Ser. No.09/668,072, filed Sep. 22, 2000 now U.S. Pat. No. 6,747,126, which is acontinuation-in-part of U.S. application Ser. No. 09/364,497, filed Jul.30, 1999 now U.S. Pat. No. 6,818,740, and which claims the benefit ofU.S. Provisional Application No. 60/132,295, filed May 3, 1999, U.S.Provisional Application No. 60/101,058, filed Sep. 18, 1998, U.S.Provisional Application No. 60/100,265, filed Sep. 14, 1998, and U.S.Provisional Application No. 60/094,676, filed Jul. 30, 1998.

This application is also a continuation of U.S. application Ser. No.09/364,497, filed Jul. 30, 1999 now U.S. Pat. No. 6,818,740, whichclaims the benefit of U.S. Provisional Application No. 60/132,295, filedMay 3, 1999, U.S. Provisional Application No. 60/101,058, filed Sep. 18,1998, U.S. Provisional Application No. 60/100,265, filed Sep. 14, 1998,and U.S. Provisional Application No. 60/094,676, filed Jul. 30, 1998.

GOVERNMENT SUPPORT

Work described herein was funded, in whole or in part, by Grant NumberP01 GM56552 from the National Institutes of Health. The United StatesGovernment has rights in the invention.

The entire teachings of all of the above-referenced applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

HIV is the virus that is responsible for the worldwide AIDS epidemic.The initial stages of HIV infection involve the fusion of viral membranewith the target cell membrane, a process that injects the viral contentsinto the cellular cytoplasm. On the viral side, the molecular complexresponsible for the fusion activity contains the surface protein gp 120and the transmembrane protein gp41. It is the current hypothesis thatgp120 interacts with the proteins CD4 and coreceptor on the target cell,resulting in a conformational change that causes gp41 to insert itsamino terminus (fusion peptide region) into the target cell membrane.This structural rearrangement promotes the fusion of virus and cellularmembranes through a poorly understood mechanism.

SUMMARY OF THE INVENTION

Described herein are chimeric peptides comprising a soluble trimericcoiled-coil and all or a portion of the N-peptide region of HIV gp41.These molecules are stable, trimeric coiled-coils that inhibit HIV entryinto cells, such as human cells. Such peptides can be further assessedto demonstrate their ability to serve as potent anti-HIV therapeuticmolecules and thus, as therapeutic molecules or drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is the structural arrangement of HIV gp41. Helical regions(heptad repeats) are shown in grey and the relative position of N-(N36)and C-(C34, DP178) peptides are indicated. In the ribbon diagram of thehelical region, the N-peptides are in light grey and the C-peptides arein dark grey.

FIG. 2 is the amino acid sequences of IQ peptides (SEQ ID Nos: 1–9).

FIGS. 3A–3C show, respectively, a helical wheel representation of IQN17(FIG. 3A); the CD spectrum of IQN17 (FIG. 3B); and analyticalultracentrifugation data for IQN17 (FIG. 3C). “XLA” is referred toherein as analytical ultracentrifugation.

FIGS. 4A–4E are photographs of results of syncytia assays carried out inthe absence of IQN peptide (FIG. 4A), in the presence of IQN17 at 80 nM(FIG. 4B) or at 320 nM (FIG. 4C) or in the presence of IQN23 at 80 nM(FIG. 4D) or at 320 nM (FIG. 4E).

FIG. 5 is a graphic representation of the inhibitory activity of IQN17and IQN23 in a viral infectivity assay.

FIG. 6 is a graphic representation of the inhibitory activity of N36 andGCN4-pI_(Q)I in a viral infectivity assay. The results presented clearlyshow a lack of inhibitory activity by both N36 and GCN-pI_(Q)I.

FIG. 7 is a working model for HIV membrane fusion (Chan & Kim 1988). Inthe native state of HIV-1 env (“Native”), the fusion-peptide andN-peptide regions of gp41 are not exposed. Following interaction withcellular receptors (CD4 and coreceptor), a conformational change resultsin formation of the transient pre-hairpin intermediate (“Pre-hairpin”),in which the fusion-peptide regions (red lines) are inserted into thecell membrane and the coiled coil of the N-peptide region of gp41(indicated as “N”) is exposed. However, the C-peptide region of gp41(indicated as “C”) is constrained and unavailable for interaction withthe coiled coil. Thus, exogenous C-peptides can bind to the pre-hairpinintermediate and inhibit fusion in a dominant-negative manner(“Inhibited”). In the absence of inhibitors, the pre-hairpinintermediate resolves to the hairpin structure and membrane fusionresults (“Hairpin/Fusion”), although it is not known if hairpinformation precedes membrane fusion per se. The inset depicts the 2.0 ÅX-ray crystal structure of N36/C34, a peptide version of the HIV-1 gp41core (Chan et al. 1997). Three central N-peptides form a coiled coil,shown here as a surface representation, and three helical C-peptidespack along conserved grooves on the surface of the coiled coil trimer.There are three symmetry-related hydrophobic pockets on the surface ofthe N-peptide coiled coil (shaded).

FIG. 8 is a graphic illustration showing that the chimeric peptides arecomposed of two parts: 1) one of the designed trimeric coiled-coils(GCN4-pI_(Q)I or IZ) (SEQ ID Nos: 10 and 11) and 2) one of the fourregions of gp41 (SEQ ID NO: 12). These regions are designated N17 (SEQID NO: 18), N23 (SEQ ID NO: 34), N36 (SEQ ID NO: 35) and N26 (SEQ ID NO:36).

FIG. 9 is a graph of concentration (nM) versus number syncytium showingthe results of the cell/cell fusion assay in the presence of IQN17 andIZN17.

FIG. 10 is the amino acid sequence of IQN26 (SEQ ID NO: 13).

FIG. 11 is the amino acid sequence of IZN26 (SEQ ID NO: 14).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to soluble peptides, referred to assoluble IQ (or IN) peptides, which, under the conditions describedherein, fold into a stable trimeric coiled-coil (helical) structure andinhibit HIV infection of mammalian cells, such as human cells. In aspecific embodiment, the soluble IQ peptides also bind D-peptideinhibitors of HIV infection of human cells, under the conditionsdescribed herein. In one embodiment, soluble IQ peptides comprise atrimeric coiled-coil peptide and a portion of the N-helix coiled-coil ofHIV gp41 (HIV gp41 N-peptide); the components are present in thefollowing “order”: N-terminus—trimeric coiled-coil peptide—N-helixcoiled coil of HIV gp41—C-terminus. The trimeric coiled-coil peptide canbe from (comprise amino acid residues that correspond to those in) avariety of sources, such as GCN4, the yeast transcription activator;Moloney Murine Leukemia Virus (MoMLV); GCN4-pII, GCN4-pI_(Q)I and theABC heterotrimer. It can also be from other designed trimeric coiledcoils such as the isoleucine zipper (IZ) described by Tanaka et al., orderivatives of this ‘IZ’ sequence. In those embodiments in which thetrimeric coiled-coil peptide is from the isoleucine zipper, they arereferred to as IZ peptides. Alternatively, it can comprise a trimericcoiled-coil peptide from HIV. Three examples of coiled-coils of interestare:

GCN4-pI_(Q)I: Ac-RMKQIEDKIEEILSKQYHIENEIARIKKLIGER-NH₂ (SEQ ID NO: 15)GCN4-pI_(Q)I′: Ac-RMKQIEDKIEEIESKQKKIENEIARIKKLIGERY-NH₂ (SEQ ID NO: 10)

-   Tanaka's isoleucine zipper: Ac-YGGIEKKIEAIEKKIEAIEKKIEAIEKKIEA-NH₂    (SEQ ID NO: 16)-   The “IZ” molecule derived from Tanaka et al., but with mutations:    Ac-YGGIKKEIEQEAIEKIAIEKEIEA-NH2 (SEQ ID NO: 11).

The amino acid residues that comprise an IQ peptide of the presentinvention can be amino acid residues that are contiguous (consecutive)or noncontiguous (nonconsecutive) in the trimeric coiled-coil peptidefrom which it is derived and/or amino acid residues that are contiguous(consecutive) or noncontiguous (nonconsecutive) in HIV gp41 N-peptide,provided that the resulting IQ peptide (the IQ peptide in which they arepresent) is stable, soluble, helical, and trimeric and inhibits HIVinfection of human cells. In the embodiments of IQ peptides in whichnonconsecutive amino acid residues of either or both components of theIQ peptide are present, the residues, as included in the IQ peptide, canbe consecutive or can be separated or joined by a linker. The linker canbe, for example, an amino acid residue(s) that do not occur between twoamino acid residues in the peptide from which the component is derived.Alternatively, the “linker” can be a chemical or synthetic linker. Acomponent of an IQ peptide of the present invention is considered to be“derived from” another peptide (e.g., a trimeric coiled-coil or HIV gp41N-peptide) if the component itself (or the nucleic acid molecule(s) thatencode the amino acid sequence) is obtained or isolated/separated from asource in which it occurs (e.g., from a cell in which the peptideoccurs, such as a portion of a protein from which it can be removed) oris produced by recombinant DNA methods, chemical synthesis or any othermethod, to comprise an amino acid sequence or a nucleic acid sequencethat is the same as or substantially the same as the sequences of thepeptide. That is, the term is intended to be interpreted broadly anddoes not require that a component be physically derived from the peptidereferred to.

In the embodiments in which the soluble IQ peptides comprise an IQregion that is a GCN4 trimeric coiled-coil peptide, they are referred toas IQN peptides. IQN peptides comprise all or a portion of GCN4-pI_(Q) I(formerly referred to as GCN4-pIQ in U.S. Provisional Application No.60/101,058; Eckert D. M. et al. J. Mol. Biol, 284: 859–865 (1998)) or amodified version of all or a portion of GCN4-pI_(Q)I, such as a modifiedportion that includes mutations for increased solubility, and all or aportion of the HIV gp41 N-peptide. Typically, 5 or more (e.g., 7, 8, 9or 10) amino acid residues from HIV-gp41 N-peptide up to and includingall of the residues of the N-peptide will comprise the HIV gp41component of the IQ peptides.

Soluble IQN peptides of the present invention comprise, in specificembodiments, a portion of the HIV gp41 N-peptide sufficient to bind theC-peptide (region) of HIV gp41 and a sufficient portion of the GCN4trimeric coiled-coil peptide or a modified version of the GCN4 peptidethat the resulting IQN peptide is a soluble trimeric (helical) coiledcoil. In further embodiments, IQN peptides comprise a portion of the HIVgp41 N-peptide that includes the amino acid residues which form thepocket or cavity of HIV gp41 (the pocket-comprising residues of theN-peptide). In yet further embodiments, IQN peptides do not compriseamino acid residues which form the pocket or cavity of HIV gp41. Theydo, however, comprise amino acid residues from HIV gp41. (See, forexample, IQN23, IQN36 and IQN26).

Nomenclature of IQ peptides refers to the number of amino acid residuesfrom the HIV gp41 N-peptide or a modified version of HIV gp41 N-peptidepresent in the IQ peptide. For example, 17 amino acid residues of HIVgp41 are included in the IQN17 peptide described herein. As explainedabove, the trimeric coiled coil peptide component of an IQ peptide mustbe sufficient in amino acid composition (identity and number/length) toresult, when joined to the HIV gp41 N-peptide portion, in formation of asoluble trimeric helical (coiled-coil) IQ peptide. In certainembodiments of the IQN peptides of the present invention, the trimericcoiled-coil peptide, referred to as the “GCN4 portion”, comprises atleast 15, 16, 17, 18, 19 or 20 amino acid residues of GCN4. The aminoacid residues present in the components of an IQN peptide can correspondto amino acid residues that are contiguous (consecutive) ornoncontiguous (nonconsecutive) in, respectively, the GCN4 transcriptionactivator (or GCN4-pI_(Q)I) and HIV gp41 N-peptide or a modified versionof the activator or the N-peptide, provided that the resulting IQNpeptide is an inhibitor of HIV infection of human cells, as describedherein. The IQ and IZ peptides of the present invention can be producedas a continuous peptide or as components that are joined or linked afterthey are formed. As used herein, the terms “joined” or “joined in such amanner” or “incorporated” include incorporating amino acid residues byeither approach.

For example, the GCN component of an IQN peptide can compriseconsecutive amino acid residues from GCN4-pI_(Q)I, modified, if desired(e.g., to increase solubility, as is the case in IQN17 (SEQ ID NO.: 1)).Alternatively, amino acid residues that are not consecutive in the GCN4activator (or in GCN4-pI_(Q)I), joined in such a manner that they arenonconsecutive or consecutive in the resulting GCN4 component of an IQNpeptide, can be incorporated in the IQN peptide. Similarly, the aminoacid residues of the HIV gp41 N-peptide component of an IQN peptide ofthe present invention can be amino acid residues that occurconsecutively or nonconsecutively in HIV gp41 N-peptide and can beincorporated into in IQN peptide in such a manner that they areconsecutive or nonconsecutive in the resulting peptide. In theembodiments in which nonconsecutive amino acid residues are used, theycan be separated by one or more “linker” molecules, if needed to retainthe respective functions/characteristics of the components and of theIQN peptide. For example, an amino acid residue(s) other than theresidue(s) that normally occur between two amino acid residues of GCN4or HIV gp41 N-peptide can be used to link or join the two amino acidresidues in the IQN peptide. Alternatively, the linker can be a chemicalor synthetic linker, for example. Under the conditions described herein,IQN peptides have been shown to fold into a stable structure, bindpeptide inhibitors of HIV-1 infection and inhibit HIV infection of humancells. For example, IQN17 and IQN23 have been shown to fold into stablestructures, bind D-peptides previously shown to be inhibitors of HIV-1infection and inhibit HIV infection of human cells. IQN36, as well asversions of IQN17 that are shortened in the ‘IQ’ region are alsodescribed. These shortened versions may be therapeutically advantageousbecause, for example, they are easier and less expensive to produce thanare larger peptides.

A specific embodiment of an IQN peptide is IQN17, which contains 29residues of GCN4-pI_(Q)I, including three mutations for increasedsolubility, and 17 residues of HIV; there is a one residue overlapbetween the two proteins, making the total length of the fusion protein45 residues. The sequence of GCN4-pIqI isac-MKQIEDKIEEILSKQYHIENEIARIKKLIGER (SEQ ID NO: 17). In this embodiment,the HIV Sequence is: LLQLTVWG IKQLQARIL (SEQ ID NO: 18). The sequence ofIQN17 is: ac-RMKQIEDKIEEIESKQKKIENEIARIKK LLQLTVWGIKQLQARIL-am (SEQ IDNO.: 1). In the sequences presented, ac represents an N-terminal acetylgroup and am represents a C-terminal amide group. IQN17 has been shownto inhibit HIV of human cells, as described herein.

Shortened versions of IQN17, which each contain 17 amino acid residuesof HIV gp41 N-helix (SEQ ID NO.: 2), but include a shorter GCN componentthan is present in IQN17, are also the subject of this invention.Specific examples of these shortened IQN17 peptides are:

-   a) shortened IQN17 #1, (SEQ ID NO.: 5), in which there are eight    amino acid residues of GCN4-pI_(Q)I: EIARIKKL (SEQ ID NO.: 19);-   b) shortened IQN17 #2 (SEQ ID NO.: 6), in which there are 15 amino    acid residues of GCN4-pI_(Q)I: KQKKIENEIAAIKKL (SEQ ID NO.: 20) and-   c) shortened IQN17 #3 (SEQ ID NO.: 7), in which there are 15 non-HIV    amino acid residues KIKKIENEIARIKKL (SEQ ID NO.: 21). This is    GCN4-pI_(Q)I′ with an I to Q mutation, and is referred to as    GCN4pII′.-   d) shortened IQN17 #4 (SEQ ID NO.: 8), in which there are 21 amino    acid residues of GCN4-pI_(Q)p: KIEEIESKQKKIENEIARIKKL (SEQ ID NO.:    22 and-   e) shortened IQN17 #5 (SEQ ID NO.: 9), in which there are 21 non-HIV    amino acid residues: KIEEIESKIKKIENEIARIKK (SEQ ID NO.: 23).

Another specific embodiment of this invention is IQN23. One embodiment,referred to as IQN23 version 1, has the following sequence:ac-RMKQIEDKIEEILSKQYHIENEIARIKKLIEAQQHLLQLTVWGIKQLQARIL-am (SEQ ID NO.:2). In IQN23 version 1, there are 29 amino acid residues in the GCN4component and 23 amino acid residues in the HIV gp41 component (a totalof 52 amino acid residues). A second embodiment of an IQN23, referred toas IQN23 version 2, also includes 23 amino acid residues of N-peptide ofgp41 and 29 amino acid residues in its GCN component, but differs fromIQN23, version 1 at amino acid residues 17 and 18 (and the L at position15 is changed to E). In version 1, these two residues are, respectively,Y and H and in version 2, they are, respectively, both K. Threemodifications have been made—the L, Y and H. These are the same threemodifications that were made to make IQN17 more soluble. The two“versions” are referred to as GCN4-pI_(Q)I and GCN4-pI_(Q)I′. Thesequence of IQN23 version 2 is:ac-RMKQIEDKIEEIESKQKKIENEIARIEAQQHLLQLTVWGIKQLQARILNH2 SEQ ID NO.: 3).As described herein, IQN23 (both versions) inhibits HIV infection moreeffectively than does IQN17.

Another specific embodiment of IQN peptides of this invention is IQN36,in which there are 30 amino acid residues of GCN4-pI_(Q)I and 36 aminoacid residues of HIV. The sequence of IQN36 (SEQ ID NO.: 4) is shown inFIG. 2. The sequence of the GCN4-pI_(Q)I component is:ac-RMKQIEDKIEEIESKQKKIENEIARIKKLI (SEQ ID NO.: 24) and the HIV aminoacid residues are: SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL—NH2 (SEQ ID NO.:25). The sequence of IQN36 is: ac-RMKQIEDKIEEIESKQKKIENEIARIKKLISGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL—NH2 (SEQ ID NO.: 3).

A wide variety of fusion proteins which are variants of IQN17 can beproduced and used to inhibit HIV. Fusion proteins include proteins madeas a single continuous molecule or as components that are subsequentlyjoined or linked together. Any of a wide variety of variations can bemade in the GCN4-pIqI component of IQN17 and used in the method,provided that these changes do not alter the trimeric state of thecoiled-coil. For example, the amino acid composition of the GCN4component can be changed by the addition, substitution, modificationand/or deletion of one or more amino acid residues, provided that thetrimeric state of the coiled-coil is maintained. For example, the Aspresidue in IQN17 (at an “f-position” of the coiled coil) can be replacedby any of the naturally-occurring amino acids. (O'Neil and DeGrado,Science 250:646 (1990)). Alternatively, this component of the fusionprotein can be a trimenic version of the coiled-coil region of anotherprotein, such as that from Moloney Murine Leukemia Virus (Fass, D. etal. Nature Struct. Biology, 3:465 (1996)), GCN4-pII (Harbury et al.,Nature, 317:80, 1994) or the ABC heterotrimer (Nautiyal and Alber,Protein Science 8:84 (1999)), or the isoleucine zipper described byTanaka, et al.

Changes can also be made in the amino acid composition of the fusionprotein component which is the C-terminal portion of the HIV gp41 Npeptide to produce a variety of fusion proteins to be used to preventHIV infection of cells. The C-terminal portion can be changed by theaddition, substitution, modification and/or deletion of one or moreamino acid residues. The amino acid composition of either or bothcomponents of the fusion protein can be altered, and there is no limitto the number or types of amino acid residue changes possible, providedthat the trimeric state of the coiled-coil is maintained. It is notnecessary that the pocket or cavity of gp41 be included, although inmany embodiments the pocket is present.

In all embodiments, controlled or time release (gradual release, releaseat a particular time after administration or insertion) of the drug canbe effected by, for example, incorporating the drug into a compositionwhich releases the drug gradually or after a defined period of time.Alternatively, the drug can be incorporated into a composition whichreleases the drug immediately or soon after its administration orapplication (e.g., into the blood, vagina, mouth or rectum). Combinedrelease (e.g., release of some of the drug immediately or soon afterinsertion, and over time or at a particular time after insertion) canalso be effective (e.g., by producing a composition which is comprisedof two or more materials: one from which release or delivery occursimmediately or soon after insertion and/or one from which release ordelivery is gradual and/or one from which release occurs after aspecified period). For example, a drug or drugs which bind the HIVcavity can be incorporated into a sustained release composition such asthat taught in U.S. Pat. No. 4,707,362. The cream, foam, gel orsuppository can be one also used for birth control purposes (e.g.,containing a spermicide or other contraceptive agent), although that isnot necessary (e.a., it can be used solely to deliver the anti-HIV drug,alone or in combination with another non-contraceptive agent, such as anantibacterial or antifungal drug or a lubricating agent). An anti-HIVdrug of the present invention can also be administered to an individualthrough the use of a contraceptive device (e.g., condom, cervical cap,diaphragm) which is coated with or has incorporated therein in a mannerwhich permits release under conditions of use a drug or drugs which bindthe HIV gp41 N-helix coiled coil. Release of the drug(s) can occurimmediately, gradually or at a specified time, as described above. As aresult, they make contact with and bind HIV and reduce or prevent viralentry into cells.

Fusion proteins of the present invention comprise a soluble, trimericform or version of a coiled-coil, such as a soluble, trimeric form orversion of a coiled-coil region of a protein (of non-HIV origin or ofHIV origin) and a sufficient portion of the C-terminal end of theN-peptide of HIV gp41 to bind to the C-peptide region. In oneembodiment, the portion of the C-terminal end of the N-peptide comprisessufficient amino acid residues to bind to the C-peptide region andinclude the HIV coiled-coil cavity or hydrophobic pocket (the pocketcomprising residues of the N-peptide). The N-peptide of HIV gp41 can bethat of HIV-1, HIV-2, another HIV strain or a strain from anotherspecies (e.g., simian immunodeficiency virus (SUV), felineimmunodeficiency virus or Visna virus). For example, HIV-2 sequenceLLRLTVWGTKNLQARVT (SEQ ID NO: 26), SIV sequence LLRLTVWGTKNLQTRVT (SEQID NO: 27) or a sequence comprising invariant residues in HIV-1, HIV-2and SIV (represented LLXLTVWGXKXLQXRXX (SEQ ID NO: 28), wherein aminoacid residues L, T, V, W, G, K, Q, and R are the single letter code usedfor amino acid residues and X can be any amino acid residue). Also thesubject of this invention is a soluble trimeric model of the HIV gp41hydrophobic pocket, which can be a D-peptide or an L-peptide andcomprises a soluble trimeric coiled-coil and a sufficient portion of theN-peptide region of HIV gp41 to comprise the amino acid residues whichform the pocket of the N-helix coiled-coil region of HIV gp41. The D- orL-peptide can comprise, as the soluble, trimeric coiled-coil, thecoiled-coil of GCN4-pI_(Q)I; GCN4-pII; Moloney Murine Leukemia Virus orthe ABC heterotrimer. The component which is a sufficient portion of theN-peptide of HIV gp41 to comprise the amino acid residues of the pocketcan comprise, for example: LLQLTVWGIKQLQARIL of HIV-1(SEQ ID NO: 18);LLRLTVWGTKNLQARVT of HIV-2 (SEQ ID NO: 26); LRLTVWGTKNTLQTRVT of SIV(SEQ ID NO: 27) or the invariant residues of these, which are:LLXLTVWGXKXLQXRXX (SEQ ID NO: 28).

One embodiment of the instant invention is fusion proteins in which thecomponents are a trimeric version of the coiled-coil region of a protein(such as GCN4-pI_(Q)I) and the N-helix coiled-coil of HIV gp41 thatinclude all, part or none of the N-helix cavity. That is, a fusionprotein of the present invention can comprise a trimeric form of thecoiled-coil region of GCN4-pI_(Q)I and a portion of the N-peptide ofHIV-1 gp41, wherein the portion of the N-peptide of gp41 comprises part,or all, or none of the N-helix cavity of HIV-I gp41. For example, afusion protein can be made that contains residues from GCNL4-pI_(Q)I andresidues from N36. The fusion protein, denoted IQN24n, contains 29residues of GCN4-pI_(Q)I, including three mutations for increasedsolubility, and 24 residues from the N-terminal end of N36(SGIVQQQNNLLRAIEAQQHLLQLT) (SEQ ID NO: 29); for recombinant expressionin E. coli, an extra Met residue is included at the N-terminus. Forexample, a fusion protein can comprise a portion of the N-peptide of HIVgp41 comprising the amino acid sequence of (SEQ ID NO: 29). The sequenceof IQN24n is: MRMKQIEDKIEEIESKQKKIENEIARIKKLISGIVQQQNNLLRAIEAQQHLLQLT(SEQ ID NO: 30). This fusion protein can be made by a variety ofmethods, including chemical synthesis or recombinant DNA methods or byrecombinant expression in E. coli, in which case the N- and C-terminiare not blocked. Because the superhelix parameters of the GCN4-pIQIcoiled-coil are nearly identical to the HIV gp41 N-helix coiled-coil,the resulting fusion protein molecule IQN24n is predicted to form a longtrimeric coiled-coil, which presents part of the gp41 N-helixcoiled-coil as a trimer (not aggregated).

The same strategy described herein to solve this problem for the gp41hydrophobic pocket can be applied towards the development of soluble,trimenic models of the gp41 N-helix coiled-coil region in general. Suchtrimeric models (including IQN 17, but also including, for example,peptides that do not contain the pocket residues of gp41) can be used asinhibitors.

Any of a wide variety of variations can be made in the GCN4-pIQIcomponent of fusion proteins described herein (e.g., IQN17 or IQN24n)and used in the method, provided that these changes do not alter thetrimeric state of the coiled-coil. Changes can also be made in the aminoacid composition of the fusion protein component which is the portionfrom the HIV gp41 N36 peptide, to produce variants (e.g., variants ofIQN17 or IQN24n). There is no limit to the number or types of amino acidresidue changes possible, provided that the trimeric state of thecoiled-coil and the structure of the surface of the fusion proteincorresponding to the N-peptide coiled coil of HIVgp41 are maintained.The fusion protein component which is the portion of the HIV gp41peptide can include all, part, or none of the N-helix cavity. Forexample, other parts of N51, N36, DP-107, or other regions of the HIVgp41 N-helix region can be fused to GCN4-pI_(Q)I (or another trimericversion of the coiled-coil region of a protein) to generate trimeric(not aggregated) helical coiled-coil fusion proteins and used in themethod. There is no limit to the number or types of fusion proteins thatcan be designed and generated, provided that the trimeric state of thecoiled-coil and the structure of the surface of the fusion proteincorresponding to the N-peptide coiled coil of HIV gp41 are maintained.Such fusion proteins can be designed and generated using methods knownto those of skill in the art, such as evaluating heptad-repeat positionsor superhelix parameters of coiled coils.

IQN17 is useful as an anti-HIV therapeutic agent, a prophylactic agentor drug to prevent HIV infection, a reagent for identification(screening for) or designing other anti-HIV therapeutics orprophylactics, and an immunogen to elicit antibodies that prevent HIVinfection.

Applicants have shown that a portion of the N-peptide can be solublizedby addition of a soluble, trimeric coiled-coil, GCN4-pI_(Q)I. Theresulting molecule is stable under physiological conditions and iscorrectly folded such that IQN17 presents a surface that is structurallycomplementary to the C-peptide region of HIV gp41. Further, IQN17 andsimilar molecules can be assessed for their ability to bind to theC-helical region of gp41, and inhibit its function. The N-helical coreof gp41 is highly conserved (in terms of amino acid composition) andthus, it is likely that IQN17 and variants thereof will be broadlyneutralizing against a variety of clinical HIV strains and, thus, usefultherapeutically.

IQN17, which is based upon the known structure of the gp41 ectodomain,consists, in one embodiment, of three N-peptides joined to (or presentin larger molecule with) a soluble trimeric coiled-coil and arranged tofold into a substantial part of the N-helical core with peptide bidingsites of the N-peptides exposed.

IQN17 protein can be produced by a variety of methods. For example, itcan be chemically synthesized. Alternatively, it can be produced, usingknown methods and expression systems, by expressing IQN17protein-encoding DNA, which can be a single DNA that encodes the entireIQN17 protein. Alternatively, protein synthetic methods can be used toproduce IQN17 protein.

IQ(IN) peptides can have a wide variety of sequences, both in theN-helix and fused coiled-coil components, and can be comprised ofL-amino acid residues, D-amino acid residues and modified amino acidsresidues. IQN17 can include amino acid residues in addition to those ofthe helices and the fused coiled-core (e.g., to stabilize the molecule).It is likely that the IQN17 described here can be altered to enhancestability and activity. Minor changes in the fused coiled-coil and theexact borders of the N-Helix are likely to have significant effects onthe stability, yield, and activity of IQN17.

As currently constructed, IQN17 exposes a portions of three C-peptidebinding sites. A strategy for exposing longer segments of the C-peptidebinding site on IQN17 (or related molecules) involves extending theN-peptide region of IQN17.

IQN17 is useful in a variety of contexts. As described herein, IQN17 isa potent inhibitor of viral membrane fusion, and, thus, acts on thevirus before it enters the cell (unlike current practical therapy, whichacts on HIV-infected cells). IQN17 is quite soluble and has been shownto be stable under the conditions described herein. It is reasonable toexpect that its size will prevent rapid filtration in the kidney. Inaddition, IQN17 dimers can be made by disulfide crosslinking, to producea molecule filtered to a lesser extent than the IQN17 “monomer”. Thus,it is reasonable to expect that dimers have an enhanced bioavailabilitywhen compared to the C-peptides.

IQN17 prevents virus from entering cells, unlike standard therapy thattargets viral proteins after viral entry, and thus, IQN17 can be usedprophylactically to prevent infection or reduce the extent to whichinfection occurs. One use for such a therapeutic is in the event of aneedlestick injury, such as might occur in a hospital or in settings inwhich needles contaminated with HIV are shared.

In one embodiment of the present invention, IQN17 is used to reduce HIVinfection in an individual. In this embodiment, IQN17 is administered,either as IQN17 itself or via expression of IQN17-encoding DNA inappropriate host cells or vectors, to an individual in sufficientquantity to reduce (totally or partially) HIV infection of theindividual's cells. That is, a dose of IQN17 sufficient to reduce HIVinfection (an effective dose) is administered in such a manner (e.g., byinjection, topical administration, intravenous route that it inhibits(totally or partially) HIV entry into cells. In one embodiment, a genetherapy approach is used to provide the effective dose, by introducingcells that express IQN17 protein into an individual. IQN17 can beadministered to an individual who is HIV infected, to reduce furtherinfection, or to an uninfected individual, to reduce infection.

The serum stability of IQN17 can be tested, using known methods toascertain its therapeutic potential.

The outside surface of the fusion coiled-coil of IQN17 can be varied,for example, to enhance bioavailability, decrease toxicity, and avoidimmune clearance. IQN17 exhibits potent inhibitory activity andGCN4-pI_(Q)I does not, it is the exposed N-peptide region that isresponsible for inhibition. The rest of the molecule provides a scaffoldfor displaying the N-peptide. Therefore, this scaffold can be modifiedwithout adversely affecting the inhibitory activity of IQN17.Modification of the scaffold may provide several advantages. First, itwould facilitate procedures in which multiple administrations of IQN17are required. For example, when IQN17 is used as an anti-HIV therapeuticagent, multiple doses might be required. After extended administration,individuals might develop antibodies to IQN17 which are likely toincrease its clearance from the body. The availability of multipleversions of IQN17 would help to circumvent this problem by evadingpreexisting antibodies. Second, it may be possible to design versions ofIQN17, for example by introducing glycosylation sites on the externalsurface, in which the scaffold is less immunogenic.

The trimer of helical hairpins (TOH) is a common feature of many viralmembrane fusion proteins (Singh, M. et al. J. Mol.Biol.290,1031–1041(1999)). It has been observed in crystal structures ofinfluenza, Ebola SV5 (simian parainfluenza virus 5), and RSV (humanrespiratory syncitial virus). In addition, many other members of theretrovirus, paramyxovirus, and filovirus families are predicted tocontain this motif. A similar structure has been observed in theassociated vertebrate vesicle fusion proteins and may be found insperm-egg, fertilization proteins. The basic strategy described hereincan be applied to any of these systems in order to inhibit fusion.

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

EXAMPLE 1 Assessment of the Specificity of IQN/Peptide Interaction andof Inhibition by IQN Peptide of Membrane Fusion

Assays were carried out to assess the ability of IQN17 to interact withthe C-region of gp41 and inhibit function of the fusion protein. Thisinhibition of membrane fusion by IQN17 and IQN23 and GCN4-pI_(Q)I wasassessed using a cell based assay. Proteins IQN17, IQN23 andGCN4-pI_(Q)I are serially diluted in modified DMEM media with 5% FCS andaliquoted into slide chambers. HELA cells (4×10⁴) expressing CD4 andcoreceptor and containing a βgalactosidase gene under the control of theTat promoter are added. CHO cells (2×10⁴) expressing gp160 (precursorprotein to gp120/gp41) and Tat are also added. The 400 μl miniculture isincubated at 37° for 8 to 24 hours; fused cells (syncytia) willtranscribe and translate β-galactosidase. The cells are fixed ingluteraldehyde and exposed to X-gal/Fe solution for one hour. Syncytiathat contain P-galactosidase turn blue-green. In this assay, IQN17demonstrates a potent inhibition of syncytia formation, with an IC₅₀ of20–80 nM.

The inhibitory potentials of IQN17, IQN23 and GCN4-pI_(Q)I have beenreproduced in viral fusion experiments. HIV, modified to contain aluciferase reporter gene, is mixed with HOS cells expressing CD4 andcoreceptor in the presence of diluted protein for 6 hours at 37° C.(Chan et al., Cell, 93, 681–684 (1998)). The virus solution is replaced,and the HOS culture is incubated 48 hours more in fresh media.Luciferase activity is measured in a luminometer. In this assay, IQN17inhibits luciferase activity with an IC₅₀ of approximately 250 nM; IQN23with an IC₅₀ of approximately 80 nM. Again, GCN4-pI_(Q)I shows noappreciable block up to ˜10 μM.

EXAMPLE 2 Assessment of Inhibition of Infectivity

Materials and Methods

Peptide Synthesis and Purification. All peptides were chemicallysynthesized on a PE Biosystems 431A peptide synthesizer upgraded withfeedback monitoring. The standard Fmoc/HBTU chemistry (Fields et al.,1991) was modified with DMSO/NMP resin swelling and acetic anhydridecapping after every couple. The peptides were cleaved from the PEBiosystems Pal resin with Reagent K. Each peptide has an acetylated Nterminus and a C-terminal amide.

The sequence of IQN17 is as described previously (Eckert et al.):Ac-RMKQIEDKIEEIESKQKKIENEIARIKKLLQLTVWGIKQLQARIL-NH₂ (SEQ ID NO: 1). Thefirst 29 residues are a non-natural designed trimeric coiled-coil, andthe final seventeen residues are derived from the N-peptide region ofHXB2 gp41 (underlined). Sequential heptads were removed from theN-terminus of IQN17 to yield three increasingly shorter peptides:IQ₂₂N17 (Ac-KIEEIESKQKKIENEIARIKKLLQLTVWGIKQLQARIL-NH₂) (SEQ ID NO: 8),IQ₁₅N17 (Ac-KQKKIENEIARIKKLLQLTVWGIKWLWARIL-NH₂) (SEQ ID NO: 6) andIQ₈N17 (Ac-EIARIKKLLQLTVWGIKQLQARIL-NH₂) (SEQ ID NO: 5). More stableversions of the IQ₂₂N17 and IQ₁₅N17 peptides were made by changing theglutamine residues to isoleucines. These peptides are called II₂₂N17 andII₁₅N17, respectively. Also, longer IQN17 derivatives were made byinserting additional residues from the HXB2 gp41 N-peptide region,taking care to keep the coiled-coil register in tact. These peptides areIQN23 (Ac-RMKQIEDKIEEIESKQKKIENEIARIKKLIEAQQHLLQLTVWGIKQLQARIL-NH₂) (SEQID NO: 2), IQN36 (Ac-RMKQIEDKIEEIESKQKKIENEIARIKKLISGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH₂) (SEQ ID NO: 4), and IQN26(Ac-RMKQIEDKIEEIESKQYKIENEIARIKKLIVQARQLLSGIVQQQNNLLRAIEAQQH-NH₂ (SEQ IDNO: 13). Finally, an additional derivative of IQN17 was made in which anentirely different designed trimeric coiled-coil was placed N-terminalto the gp41-derived residues. The coiled-coil was based on a designdescribed by Tanaka et al., but has significant alterations in the e andg positions and an I to Q substitution at an a position. The sequence ofthis peptide, called IZN17, is Ac-IKKEIEKKEQEAIKKKIEAIEKLLQLTVWGIKQLQARIL-NH₂ (SEQ ID NO: 31). Additional peptides that arebeing studied for their inhibitory activity are: IZN23(Ac-IKKEIEAIKKEQEAIKKKIEKEIEAQQHLLQLT VWGIKQLQARIL-NH₂ID NO: 32), IZN36(Ac-IKKEIEAIKKEQEAIKKKIEAIEKEISGIVQQQNNLLRAIEAQQHLLQLTVWGIKQL QARIL-NH₂)(SEQ ID NO: 33) and IZN26:(Ac-YGGIKKKEIEAIKKEQEAIKKKIEAIEKEIVQARQLLSGIVQQQNNLLRAIEAQQH-NH₂ (SEQ IDNO: 14).

Following cleavage from the resin, each peptide was desalted over aSephadex G-25 column (Pharmacia) and lyophilized. It was thenresuspended in 5% acetic acid and purified over a Vydac C18 preparativecolumn on a reverse phase high-performance liquid chromatographyapparatus (Waters, Inc.). The peptide was eluted from the column with awater-acetonitrile gradient in the presence of 0.1% trifluoroacetic acidand then lyophilized. The molecular weights of each peptide werevalidated using MALDI-TOF mass spectrometry (PerSeptive Biosystems).

Circular Dichroism. All CD measurements were performed on an Aviv 62 DScirucular dichroism spectrometer. Standard scans were performed on 10 μMsolutions of peptide in PBS (50 mM sodium phosphate, 150 mM sodiumchloride [pH 7.4]) from 200 to 260 nm in a 1 cm pathlength cuvette witha 5 second averaging time. The mean residue ellipticity (θ) wascalculated by dividing the raw signal by peptide concentration (M),pathlength (mm) and number of amino acids. Percent helicity wascalculated according to Chen et al. (Biochemistry, 13, 1974, p3350).Thermal denaturation scans of 10 μM peptide solutions in PBS wererecorded at 222 nm. The peptide was heated at two degree intervalsstarting at 4° C., with an equilibration time of 1.5 minutes and anaveraging time of 60 seconds.

Sedimentation Equilibrium. All measurements were recorded on a BeckmanXL-A analytical ultracentrifuge equipped with an An-60 Ti rotor.Lyophilized peptide was resuspended in water, and the peptideconcentration was determined (Edelhoch, 1967). The solution was dilutedto 100–200 μM and then dialized overnight against PBS. Followingdialysis, the concentration was redetermined and the appropriatedilutions were made, using the dialysis buffer. The samples werecentrifuged at speeds ranging from 19,000 to 25,000 RPM.

HIV Infectivity Assay. Inhibitory activity of IQN17 and derivatives wasdetermined in an HIV luciferase assay (Chen et al., 1994). Specifically,virus was made by cotransfecting 293T cells with an HIV-1 genomecontaining a frame-shift mutation in env and luciferase replacing thenef gene (NL43LucR-E-) along with pCMVHXB2, an expression vector withthe HXB2 gp160 gene. The resultant virus is only viable for one round ofinfection since its genome lacks the envelope gene. The cellular debriswas removed by low-speed centrifugation. The remaining viral supernatantwas used to infect HOS-CD4/Fusion cells (N. Landau, National Institutesof Health AIDS Reagent Program) in the presence of the potentiallyinhibitory peptides. Two days post infection, the cells were lysed andluciferase activity was monitored on a Wallac AutoLumat LB953luminometer (Gaithersburg, Md.). IC₅₀s (the peptide concentration atwhich half of the viral infection is inhibited) were calculated byfitting the data to a Langmuir equation [y=k/(1+[peptide]/IC₅₀)], wherey=luciferase activity and k is a scaling constant.

Results

The pocket-forming region of the N peptide inhibit as a coiled-coiltrimer. The x-ray crystal structure of fusogenic gp41 shows acoiled-coil trimer of N peptides surrounded by three helical C peptides.A hydrophobic pocket at the base of the N peptides, into which threehydrophobic side chains from the each of the C peptides pack, has beenshown to be an important target for anti-HIV-1 compounds. IQN17 is achimeric molecule designed to accurately present this hydrophobic pocketin its proper trimeric coiled coil conformation, in the absence of Cpeptides (Eckert et al.). A designed trimeric coiled coil,GCN4-pI_(Q)I′, was fused to the N-terminus of seventeen residues of theN-peptide. These seventeen residues span the pocket region of the Npeptide. Coiled coils are composed of a characteristic repeat of sevenresidues (designated a through g), with the first (a) and fourth (d)positions typically occupied by hydrophobic side chains. Carefulattention was taken to fuse the GCN4 portion and the N-peptide portionin proper coiled coil register. Here, we assayed the inhibitory activityof this chimeric molecule, and determined that this activity is relianton the coiled coil conformation.

Two synthetic peptides were compared: N17, containing the seventeenresidues of the N peptide that comprise the hydrophobic pocket, andIQN17. N17 is difficult to get into solution—it precipitates. Thereforeit is unlikely to be a discretely trimeric coiled coil. Alternatively,IQN17 is easily dissolved. It is a fully helical and discretely trimericspecies at 20 μM. IQN17 is extremely stable, with a melting temperatureabove 100° C. Both peptides were assayed for their ability to inhibitviral infection. N17 does inhibit infection, but has an IC₅₀ ofapproximately 10 μM. IQN17 inhibits infectivity at approximately twoorders of magnitude lower concentrations, with an IC₅₀ of approximately180 nM. Therefore, the inhibitory activity of the N17 region of the Npeptide is greatly enhanced in a trimeric coiled-coil conformation.

The pocket of IQN17 contains the inhibitory activity. To rule out thepossibility that the IQ-portion of IQN17 is responsible for all or someof the inhibitory activity, two control molecules were studied. Thesepeptides are GCN4-pI_(Q)I′ and IQN17 (G572D). GCN4-pI_(Q)I′ consists ofonly the IQ portion of IQN17. IQN17 (G572D) contains a mutation in thelining of the hydrophobic pocket. A glycine residue is changed toaspartate, introducing a charge into the otherwise hydrophobicenvironment. Both of these molecules are helical as determined bycircular dichroism, and therefore serve as proper controls,structurally. In infectivity assays, they have little, if any inhibitoryactivity. GCN4-pI_(Q)I′ does not inhibit at any concentrations tested(so far up to 10 μM), and the IC₅₀ of the IQN17 (G572D) is about 20 μM.Therefore the hydrophobic pocket of IQN17 is responsible for theinhibitory activity of IQN17, with the IQ portion likely serving topresent the pocket in the proper conformation.

The inhibitory potency of IQN17 is correlated to its stability. A seriesof peptides were studied to determine how much of the IQ portion of themolecule is required for the coiled coil structure and inhibitoryactivity of IQN17. Groups of seven residues were sequentially removedfrom the N-terminus of IQN17 to make peptides of 38, 31 and 24 aminoacids in length (IQ₂₂N17, IQ₁₅N17 and IQ₈N17, respectively). IQ₂₂N17showed a characteristic alpha helical CD spectrum at 10 μM with aminimum of approximately −36,000 deg cm² dmol⁻¹ at 222 nm. Notsuprisingly, it is much less stable than wild type IQN17, with a meltingtemperature below 80° C. at 10 μM (IQN17 has a melting temperature above100° C. at the same concentration). IQ₁₅N17, which is seven residuesshorter than IQ₂₂N17, is slightly less helical with a minimum ofapproximately −27,000 deg cm² dmol⁻¹ at 222 nm. Also, the minimum at 208nm is slightly lower than that at 222 nm, implying a partial unfoldingof this peptide. Its thermal stability is about 10 degrees lower thanthat of IQ₂₂N17. Finally, circular dichroism studies of IQ₈N17 show amuch lower minimum at 208 than at 222 nm, implying that this peptide isnot very helical. It is also much less stable than the first twopeptides. The inhibitory activity of each of these peptides issignificantly lower than that of wild type IQN17, despite the fact thatIQ₂₂N17 is as helical as IQN17 at 10 μM. The IC₅₀ of IQ₂₂N17 for viralinfectivity is around 1 μM, and the IC₅₀s of the two shorter moleculesis close to 10 μM (very similar to the inhibitory activity of N17).Since IQ₂₂N17 is much less stable than IQN17, it is likely that it isunfolded at the lower concentrations in which IQN17 demonstratesinhibitory potency.

To determine if it is lowered stability that has negatively affected theinhibitory activity of IQ₂₂N17 and IQ₁₅N17, two additional peptides weremade in which the glutamine in the a position of the IQ portion of theabove two molecules was mutated to isoleucine. Previously it had beenshown that mutating an isoleucine to a glutamine in the core of atrimeric coiled coil drastically reduces the stability of the coiledcoil (Eckert, Malashkevich, Kim). These additional peptides are calledII₂₂N17 and II₁₅N17. II₂₂N17 is extremely helical at 10 μM, with anapproximate 222 nm minimum of −41,000 deg cm² dmol⁻¹. II₁₅N17 isslightly less helical with an approximate −33,000 deg cm² dmol⁻¹minimum. As with IQ₁₅N17, the 208 nm minimum is slightly lower than the222 nm minimum. The stability of these peptides and their inhibitoryactivity is increased relative to the glutamine versions. At 10 μM,II₂₂N17 is not melted even at 100° C. and its IC₅₀ is approximately 170nM. II₅N17 is more stable than IQ₁₅N17, and its IC₅₀ is approximately 3μM. Therefore it seems likely that the inhibitory activity of thepeptides is correlated to the stability of the coiled-coil structures.

Increasing the length of the N peptide region does not necessarilyincrease inhibitory potency. Since IQN17 has a binding site for the Cpeptide region of gp41, it likely inhibits by binding to this regionduring the process of viral membrane fusion. Therefore, it is possiblethat by extending the length of the N-peptide region, thereby increasingthe C peptide binding area, the inhibitory activity will improve. Totest this hypothesis two additional peptides, IQN23 and IQN36, wereconstructed. They have seven and 19 additional residues from gp41,N-terminal to the pocket region, respectively. Analyticalultracentrifugation studies show that these peptides are more aggregatedthan IQN17 (with Mobs/Mcalc of 3.3 for IQN23 and 3.5 for IQN36 at 20μM). The IC₅₀ of IQN23 is about 30 nM, and IQN36 has an IC₅₀ ofapproximately 50 nM. N36, just the gp41 region of IQN36, has an IC₅₀ ofaround 1 μM, as comparison. By increasing the gp41 residues from 17 to23, there was an approximate 6-fold gain in inhibitory activity.Likewise, by increasing the residues from 17 to 36, there is a 3-foldgain. However, IQN23 is more potent than IQN36. Therefore, theinhibitory activity does not increase just due to adding N peptideresidues. There is likely a trade-off between C-peptide binding energyand aggregation state. The longer the N peptide region is, the moreaggregated the molecule is, and it therefore more poorly represents adiscretely trimeric C peptide binding site.

The pocket region of the N peptide is not required for inhibitoryactivity. To determine if the pocket region was required for theinhibitory potency of the chimeric N peptide molecules, an additionalpeptide was made, IQN26. This peptide contains 26 residues of the Npeptide region N-terminal to the hydrophobic pocket. Circular dichroismstudies show it is helical, and sedimentation equilibrium studies showit is slightly aggregated. It does have potent inhibitory activity.Therefore, the N peptide region, when constrained in a coiled coilformation, has inhibitory activity even in the absence of thehydrophobic pocket region.

An alternate, more stable peptide, IZN17, is a more potent inhibitor. Westudied an additional IQN17 derivative, in which the IQ portion of themolecule was replaced with another trimeric designed coiled coil. Thiscoiled coil, called ‘IZ’ for isoleucine zipper, is based on a designdescribed by Tanaka, et al., but has several changes in the e and gpositions and an isoleucine to glutamine substitutation at an aposition. The resulting peptide is termed IZN17. IZN17 is helical anddiscretely trimeric at 20 μM as determined by circular dichroism andsedimentation equilibrium, respectively. Interestingly, IZN17 has anIC₅₀ of approximately 5.6 nM in the viral infectivity assay, and istherefore a much better inhibitor than IQN17. There are two potentialreasons for this increase in potency. First, IZN17 is likely more stablethan IQN17, and therefore stays folded at lower concentration. Bothpeptides melt above 100° C., although thermal unfolding transitions canbe seen in the presence of denaturant. In 2 M GuHCl, the thermaldenaturation temperature of IZN17 is ten degrees higher than that ofIQN17. Second, IZN17 contains two additional residues from the gp41 Npeptide region, due to a coincidence in sequence between IZ and gp41.This could provide an increase in binding energy to the C peptide regionof gp41.

The inhibitory activity of IZN23, IZN36 and IZN26 can be tested usingknown methods, such as those described herein. These peptides are likelyto be potent inhibitors of HIV-1 infection.

The following Table summarizes the biophysical data and inhibitoryactivity for the chimeric coiled coil N peptides. The first column (θ₂₂₂nm) is circular dichroism data, representing the helicity of eachpeptide. The lower the number, the more helical. The second column isthe melting temperature and signifies the temperature at which half ofthe peptide is unfolded. The next column (M_(obs)/M_(calc)) signifiesthe oligomeric state of the peptide, with 3.0 being a discrete trimer.The final column is the concentration at which each peptide is at halfmaximal inhibitory potency for viral infection.

TABLE 1 Biophysical data and HIV-1 inhibitory activity of IQN17 andderivatives. θ_(222 nm) Melting (deg cm² Temperature M_(obs)/ PEPTIDEdmol⁻¹) (° C.) M_(calc) IC₅₀ (nm) IQN17 ~−36,000 >100 3.15  ~180 N17n.d. n.d. n.d. ~10,000 GCN4-pI_(Q)I’ ~−31,000 68 3.00  >10,000IQN17(G572D) ~−39,000 n.d. n.d. ~20,000 IQ₂₂N17 ~−36,000 ~76 2.73*~1,000 IQ₁₅N17 ~−27,000 ~64 n.d. ~10,000 IQ₈N17 n.d. n.d. n.d. ~10,000II₂₂N17 ~−41,000 >100 3.02  ~170 II₁₅N17 ~−33,000 ~76 2.64  ~3,000 IQN23n.d. n.d. 3.29  ~30 N23 n.d. n.d. n.d. n.d. IQN36 n.d. n.d. 3.47  ~50N36 n.d. n.d. n.d. ~1,000 IQN26   ~25,000 ~70 3.25  ~43 IZN17~−31,000 >100 3.05  5.6 IZN23 n.d. n.d. n.d. n.d. IZN36 n.d. n.d. n.d.n.d. IZN26   ~33,000 ~82 ~2.87 n.d. *this was determined at 50 μMMany of these values are the results of one experiment, and aretherefore tentative. Results will need to be confirmed by repeatingexperiments. n.d. means not determinedLegend:Table 1 lists all of the peptides studied in this paper. The firstcolumn gives the name of the peptide. The second column is theellipticity at 222 nm of a 20 μM solution of peptide in PBS. The thirdcolumn lists the midpoint of thermal denaturation of 10 μM solutions ofpeptide in PBS. The fourth column gives the ratio of the observedmolecular weight of 20 μM peptide solutions in PBS by analyticalultracentrifugation to the calculated molecular weight for a monomericpeptide. The final column give the concentration of peptide at whichhalf of viral infectivity is inhibited (IC₅₀).

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A soluble trimeric coiled-coil peptide comprising a peptide IZN26,wherein the amino acid sequence of IZN26 is: (SEQ ID NO:14) Ac-Y   G   G   I   K   K   E   I   E   A   I   K    (Tyr Gly Gly Ile LysLys Glu Ile Glu Ala Ile LysK   E   Q   E   A   I   K   K   K   I   E   A   I Lys Glu Gln Glu AlaIle Lys Lys Lys Ile Glu Ala IleE   K   E   I   V   Q   A   R   Q   L   L   S   G Glu Lys Glu Ile ValGln Ala Arg Gln Leu Leu Ser GlyI   V   Q   Q   Q   N   N   L   L   R   A   I   E Ile Val Gln Gln GlnAsn Asn Leu Leu Arg Ala Ile Glu A   Q   Q   H  —CONH₂ Ala Gln Gln His).


2. A soluble trimeric coiled-coil peptide consisting of a peptide IZN26,wherein the amino acid sequence is: (SEQ ID NO:14) Ac-Y   G   G   I   K   K   E   I   E   A   I   K    (Tyr Gly Gly Ile LysLys Glu Ile Glu Ala Ile LysK   E   Q   E   A   I   K   K   K   I   E   A   I Lys Glu Gln Glu AlaIle Lys Lys Lys Ile Glu Ala IleE   K   E   I   V   Q   A   R   Q   L   L   S   G Glu Lys Glu Ile ValGln Ala Arg Gln Leu Leu Ser GlyI   V   Q   Q   Q   N   N   L   L   R   A   I   E Ile Val Gln Gln GlnAsn Asn Leu Leu Arg Ala Ile Glu A   Q   Q   H  —CONH₂ Ala Gln Gln His).